Formalin-fixed Paraffin Sections Research Articles

Keratan sulfate proteoglycan: putative template for neuroblast migratory and axonal fascicular pathways and fetal expression in globus pallidus, thalamus, and olfactory bulb.

Keratan sulfate (KS) is a proteoglycan secreted in the fetal brain astrocytes and radial glia into extracellular parenchyma as granulofilamentous deposits. KS surrounds neurons except dendritic spines, repelling glutamatergic and facilitating GABAergic axons. The same genes are expressed in both neuroblast migration and axonal growth. This study examines timing of KS during morphogenesis of some normally developing human fetal forebrain structures. Twenty normal human fetal brains from 9-41 weeks gestational age were studied at autopsy. KS was examined by immunoreactivity in formalin-fixed paraffin sections, plus other markers including synaptophysin, S-100β protein, vimentin and nestin. Radial and tangential neuroblast migratory pathways from subventricular zone to cortical plate were marked by KS deposits as early as 9wk GA, shortly after neuroblast migration initiated. During later gestation this reactivity gradually diminished and disappeared by term. Long axonal fascicles of the internal capsule and short fascicles of intrinsic bundles of globus pallidus and corpus striatum also appeared as early as 9-12wk, as fascicular sleeves before axons even entered. Intense KS occurs in astrocytic cytoplasm and extracellular parenchyma at 9wk in globus pallidus, 15wk thalamus, 18wk corpus striatum, 22wk cortical plate, and hippocampus postnatally. Corpus callosum and anterior commissure do not exhibit KS at any age. Optic chiasm shows reactivity at the periphery but not around intrinsic subfasciculi. We postulate that KS forms a chemical template for many long and short axonal fascicles before axons enter and neuroblast migratory pathways at initiation of migration. Cross-immunoreactivity with aggrecan may render difficult molecular distinction.

P11.19.A MACROPHAGES-BASED IMMUNOTHERAPY OF SOLID TUMORS MICROENVIRONMENT: THE TEM-GBM STUDY (NCT03866109)

Abstract BACKGROUND We have developed an HSC-based platform to switch from pro-tumor to anti-tumor bone-marrow derived tumor-infiltrating macrophages. Specifically, Temferon cell therapy delivers IFN-a into the tumor microenvironment (TME) in a targeted manner via Tie-2 expressing monocytes, with transcriptional and post-transcriptional controls mediated by miRNA target sequences. MATERIAL AND METHODS TEM-GBM is an open-label, Phase 1/2a dose-escalation study evaluating the safety and efficacy of Temferon in up to 27 newly diagnosed, unmethylated MGMT glioblastoma (GBM) patients. Twenty-four patients are assigned to 8 cohorts and will receive a single increasing dose of Temferon, 3 additional patients will be further enrolled to confirm the safety profile of the chosen Temferon dose to be tested in a broader group of patients (Phase 2). Patients are followed up to 2 years after treatment with Temferon. Here we report the analysis on the biological activity of Temferon on patients who underwent a second surgery intervention. RESULTS As of 31st March 2023, 6 out of 19 Temferon treated patients underwent a second surgical intervention; of these, 4 belonged to low dose cohorts (C1-C2) and 2 to high dose cohorts (C4-C5). The second surgery occurred at a median of 11 months after 1st surgery (range 7-17 months). Where enough fresh material was collected to enrich for CD45+ cells within the tumor, a 3% to 5% of gene-marked tumor infiltrate was found (2/2 specimens) suggesting that Temferon-derived cells homed to the TME. To verify if the local delivery of IFN-a by TEMs was able to upregulate the Interferon Stimulated Genes (ISGs) in tumor tissue, the transcripts for IFIT1, IRF7, OAS1a genes were evaluated by RT-PCR in formalin-fixed paraffin sections obtained at first and second surgery. The analysis was performed on eighteen (18) 1st surgery samples (from C1 to C6), and on six (6) 2nd surgery specimens. At 1st surgery, transcripts were detected in 17 out of 17 specimens (one sample excluded since housekeeping genes did not amplify) and most of the patients showed a relatively low quantity of ISGs expression. The comparison of the overall ISGs expression at first surgery and second surgery highlighted the activation of IFN-responsive genes at second surgery suggesting the occurrence of the local delivery within the TME of biologically active IFN-a. CONCLUSION The results provide initial evidence of Temferon’s potential to modulate the TME of GBM patients.

P111 Efficacy of a novel long-acting lipidated interleukin-22, alone and in combination with anti-TNF treatment, in a murine chronic DSS colitis model

Abstract Background Most current treatments and new agents in clinical development for Inflammatory Bowel Disease focus on inhibition of excessive inflammation. However, to break the ceiling on current clinical remission rates, new approaches, for example targeting mucosal healing, are needed. The cytokine interleukin-22 (IL-22) plays a key role in epithelial healing and has early proof-of-concept as a potential drug class in a phase 1 trial in Ulcerative Colitis. Here we present results from a mouse model of colitis demonstrating the efficacy of IL-22 agonism and complementarity with existing immunomodulatory treatment options. Methods Eight-week-old female C57Bl/6N mice (n=56) were subjected to a DSS-induced model of colitis; mice were given 2% (weight/volume) DSS in the drinking water for three 7-day cycles followed by 7 days of regular drinking water. Mice were treated from day 18 until the end of study with either vehicle, lipidated IL-22, an anti-murine TNF antibody at a suboptimal dose, a combination of the lipidated IL-22 and anti-TNF, and, as positive control, anti-murine TNF at an optimal dose. Colitis severity was monitored during the study and at study end endoscopic scoring was performed. Colon length and weight were measured, and histological analysis on formalin-fixed paraffin sections performed by a blinded pathologist. Results At termination, all treatment groups showed reduced body weight loss and improved DAI scores compared to vehicle treated animals, which was significant for all but the suboptimal anti-TNF group. Combination treatment resulted in an additive treatment effect compared to single treatment alone, with mean DAI improving by 13% and 48% for the anti-TNF and IL-22 groups alone versus 68% in combination. Similar effects were observed for the endoscopic and histological scores. The compositive endoscopic score improved by 21% (anti-TNF) and 55% (IL-22) versus 66% for the combination, and histological scores 22% and 52% versus 66%. These combination results likely represent the maximum response that can be achieved in the model, since the improvements obtained are the same as those for the treatment positive control for the model (68% and 62% improvements in endoscopic and histological scores, respectively). Conclusion A novel long-acting lipidated IL-22, which acts on epithelial repair mechanisms, demonstrated strong and significant improvements on all measured parameters in a chronic DSS model of colitis. Combining the IL-22 mode-of-action with an immunomodulatory agent, exemplified by a murine anti-TNF antibody, demonstrated the complementarity of IL-22 agonism to existing treatment options. Clinical trials to assess safety and tolerability of lipidated IL-22 are expected to commence at the start of 2023.

Prolactin Inducible Protein, but Not Prolactin, Is Present in Human Tears, Is Involved in Tear Film Quality, and Influences Evaporative Dry Eye Disease.

PurposeDecreased production of the aqueous component of the tear film is an important cause of the development of dry eye disease (DED). Tear production is influenced by hormones and hormone-like factors. Prolactin (PLR), a multifunctional pituitary gland hormone, is regularly present in the lacrimal gland of rats and rabbits. In humans, serum PLR concentration correlates with tear quality. To gain deeper insights of possible effects of PRL, prolactin receptor (PRLR) and prolactin inducible protein (PIP), we analyzed the three proteins in the human lacrimal apparatus and in reflex tears of healthy volunteers as well as patients suffering from DED.MethodsGene expression of PRLR and PIP was analyzed by RT-PCR in cadaveric human lacrimal gland and ocular surface tissues, immortalized human corneal epithelial cells (HCE and hTEPI) and human Meibomian gland epithelial cells (HMGECs). At the protein level, the expression and localization of PRL, PRLR and PIP in formalin-fixed paraffin sections of the lacrimal apparatus were studied by immunohistochemistry. In addition, tear fluid from DED patients and healthy volunteers was analyzed by ELISA to determine the concentration of PRL and PIP.ResultsRT-PCR analyses revealed gene expression of PRLR and PIP in human tissue samples of cornea, lacrimal glands, and eyelids, whereas only PIP, but not PRLR, was detectable in immortalized corneal epithelial cells. Immunohistochemistry revealed for the first time the expression and localization of PRL, PRLR, and PIP in human tissues of the lacrimal apparatus and at the ocular surface. PRL and PRLR were detectable in corneal epithelium, lacrimal glands, and Meibomian glands. Reflex tears from DED patients revealed significantly increased PIP concentrations, whereas PRL was undetectable in tears of DED patients and healthy volunteers.ConclusionPRL, PRLR, and PIP are found in the lacrimal apparatus and on the ocular surface. PIP, but not PRL, is present in human tears and appears to be involved in the physiology of tear film quality. Our clinical data revealed that PIP may affect tear quality, but further functional analyses are needed to fully elucidate the effects of PRL and PIP-associated factors in tear secretion as well as in the connection of DED.

Abstract 4222: Phenstatin based indole linked chalcone compound 9a exhibits anti-oral cancer activity through regulating NLRP3 inflammasome innate immune pathway

Abstract Oral cancer is the sixth most prevalent malignancy in the world and oral squamous cell carcinoma accounts for majority of all oral malignancies. Upregulated NLRP3 inflammasome innate immune pathway is of importance to tumor development. Current efforts are being focused on identifying small molecules that exhibit anti-cancer activity as inflammasome pathway inhibitors. Our previously published work on phenstatin based indole linked chalcone scaffold 9a with 1-methyl, 2- and 3-methoxy substituents in the aromatic ring revealed 9a as an anti-oral cancer compound. 9a was found to act through inhibiting tubulin polymerization at protein level, using in vitro models oral cancer cell line/spheroid cells and in vivo animal oral cancer xenograft model. 9a had also shown significant reduction in radiolabeled-glucose uptake in xenograft mice model. Current study was undertaken to evaluate if small molecule inhibitor 9a acts through regulating the NLRP3 pathway. Using computation approach, we predicted the binding of 9a with NLRP3NACHT domain, which revealed stable interaction as similar to that exhibited by NLRP3 inhibitors MCC950 and ADP. Further, we checked immune mechanistic activity of 9a on NLRP3 pathway intermediates in oral cancer cells. AW13516 cell line which was human tongue squamous tumor-derived cell line; indigenously developed at our department previously, was activated for NLRP3 inflammasome pathway using LPS and activator Nigericin in presence of 9a. MCC950 treated cells and only LPS or LPS/Nigericin treated cells served as controls. NLRP3, caspase-1 and mitochondrial protein expression was analyzed in these cells by immunofluorescence (IF) and found to be increased upon LPS/NIG activation and reduced significantly upon MCC950 and 9a treatment. Activation led to puncta formation which was found diffused after MCC950/9a treatment. Similarly treated AW13516 cells were also validated using western blotting experiments. Expression of 118kDa NLRP3 protein was found increased upon inflammasome activation that was significantly reduced in 9a treated cells and reduction was dose dependent. 9a had shown significant reduction in oral cancer xenograft of AW13516 in NOD-SCID mice model. We tested formalin-fixed paraffin sections of these tumors by immunohistochemistry. Tumor areas were assessed for expression of NLRP3 pathway markers and there was significant reduction in NLRP3, Caspase-1, GSDMD and IL-1β in 9a treated tumors compared to control tumors. This reduction was at par with that shown by Adriamycin. Summarizingly, 9a has been found to be regulating inflammatory immune mechanisms and can be developed further as immunomodulatory anti-cancer agent. Since macrophages are major resource immune cells of NLRP3, further studies are underway to test 9a on macrophages from nlrp3-/- and caspase-/- mice in comparison with normal mice. Citation Format: Jyoti Kode, Jitendra Maharana, K. Nirmal Kumar, Trupti Pradhan, Arvind Ingle, Madan Barkume, Meena Patkar, Namitha Thampi, Ankita Patil, Anand Vaibhaw, Jeshma Kovvuri, Ahmed Kamal. Phenstatin based indole linked chalcone compound 9a exhibits anti-oral cancer activity through regulating NLRP3 inflammasome innate immune pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4222.

Ground state depletion microscopy as a tool for studying microglia-synapse interactions.

Ground state depletion followed by individual molecule return microscopy (GSDIM) has been used in the past to study the nanoscale distribution of protein co‐localization in living cells. We now demonstrate the successful application of GSDIM to archival human brain tissue sections including from Alzheimer's disease cases as well as experimental tissue samples from mouse and zebrafish larvae. Presynaptic terminals and microglia and their cell processes were visualized at a resolution beyond diffraction‐limited light microscopy, allowing clearer insights into their interactions in situ. The procedure described here offers time and cost savings compared to electron microscopy and opens the spectrum of molecular imaging using antibodies and super‐resolution microscopy to the analysis of routine formalin‐fixed paraffin sections of archival human brain. The investigation of microglia–synapse interactions in dementia will be of special interest in this context.

Quantitative analysis of RNAscope staining for c-fos expression in mouse brain tissue as a measure of Neuronal Activation

The expression of c-fos mRNA is an indirect marker of neuronal activity. RNAscope ACD Bio RNAscope (now Biotechne) is a proprietary in-situ mRNA detection technology using branched DNA amplification and z paired probes to deliver a robust and specific assay designed primarily for use on formalin fixed paraffin sections [1]. In the present study we adapted this technology to be used in frozen sections to allow quantitative analysis of c-fos gene expression in different mouse brain regions during neuropharmacology studies. The method was applied by Cosi et al. 2021 [2] and the image analysis is described here in details.•The patented RNAscope (ACD Bio) flourescent in-situ hybridisation technology designed primarily for use on formalin fixed paraffin sections was adapted to be used on frozen section from mouse brain.•We carefully controlled sample preparation and handling to maximise mRNA preservation and used the fluorescent properties of the fast Red substrate combined with fluorescent whole slide scanning and image analysis.•A customized algorithm was set up for image analysis•The method developed permitted the quantitative analysis of c-fos expression in specific brain regions from whole sections.

The Correlate between Argyrophilic Nucleolar Organizer Region Stain and Ki-67 Immnochistochemistry in Diagnostic Breast Cancer

Introduction: This experimental study gauged the value of argyrophilic nucleolar organizer region (AgNOR) staining as a possible technique for the estimation of cell kinetics in conventional histology sections, in benign and malignant breast lesions. Methods: With a silver staining technique and immunohistochemistry, we associated the numbers of AgNORs and Ki67 scores in 30 breast carcinomas and 10 benign breast lesions. Results: The mean values of Ag NORs silver stain dots count for normal, benign, grade II and III were 1.28±0.17, 2.83±0.68, 5.23±0.87 and 7.32±0.92, respectively. There was a statistically significant difference (p≤0.001) was noticed between the all individual groups, among the normal and breast lesion as well as among the GII and GIII. Immunohistochemical Results of Ki-67 protein exhibited homogenous golden-brown color in control case and a positive brown granules or diffuse dark brown color in the nuclei of both benign and malignant cases under the 400X magnify examined under the light microscope. Discussion: AgNOR counts performed on routine formalin-fixed paraffin sections could provide substantial kinetic evidence. Additionally, the difference in AgNOR counts between benign and malignant tumors is such that they may be of diagnostic worth.

A study on qualitative comparison between cryostat and conventional technique for histopathology diagnosis

Introduction: Intraoperative consultation by frozen section technique is an invaluable tool for immediate diagnosis. The correlation of intraoperative frozen section diagnosis with final diagnosis on permanent section is an integral part of quality assurance in surgical pathology. Its accuracy and limitations vary with different anatomical sites. Aims & Objective: Qualitative morphological comparison between frozen section and routine formalin fixed paraffin embedded sections in different tissues, to assess the accuracy of frozen section, to detect the number and type of discrepancies and to assess the causes for discrepancies. Material and Method: The present study retrospectively reviewed frozen sections performed in the pathology department, Sir-t hospital, government medical college, Bhavnagar during a period of 2 year. Diagnostic accuracy of frozen section and its morphological quality and reliability in comparison to histopathology was evaluated by 2 pathologists in a blinded fashion for the following parameters: cellular outline, nuclear and cytoplasmic features, staining pattern and overall morphology. The results were compared to the routine formalin fixed paraffin sections to evaluate diagnostic accuracy. Discordant cases were reassessed to find the reasons for discrepancy. Result: Diagnostic accuracy of frozen section was 95.1% with false negative case are 4.8% and no false positive case. Statistical analysis showed that most common indications of frozen section in the present study was presence/typing of neoplasm (61.2%) followed by assessment of margin (35.6%) and assessment of nodal status (3.2%). It was observed that nuclear details, cellular outline and overall morphological quality of frozen section was slightly inferior to that of routine histopathology section, however, staining and cytoplasmic details were comparable. The discrepancies were mainly due to technical artefacts (9.7%), sampling error (3.2%) interpretation error (1.6%), and partly due to lack of interdepartmental communication. Conclusion: Frozen section diagnosis is very useful and highly accurate procedure. Gross inspection, sampling by pathologist, frozen complemented with cytological and histological review and intimal cooperation with surgeon can avoid certain limitations and provide rapid, reliable, cost effective information necessary for optimum patient care.

Abstract 3165: Identification of candidate biomarkers for aggressive prostate cancer using targeted proteomics and FFPE tissue samples with outcomes data

Abstract Introduction: Although many (~40%) of the screen-detected prostate cancers are indolent (Gleason Score <= 6) and pose minimal risk for progression, advanced stage prostate cancer is a lethal disease with 5-year survival rates around 29%. The challenge is to identify biomarkers for early detection of aggressive disease, when the cancer is still organ confined. Such markers would be also used to better select patients with indolent and low risk cancers for active surveillance. Material and Methods: To identify a panel of protein markers that could predict prostate cancer progression, we developed ultra-sensitive, high-pressure, high-resolution separations coupled with intelligent selection and multiplexing-selected reaction monitoring (PRISM-SRM) assays for 52 protein markers. Candidate protein markers were identified and selected from existing prostate cancer genomics data sets and validated lists of known prostate cancer drivers. The PRISM-SRM assays used heavy isotope-labeled synthetic peptides as internal standards for quantitative proteomics analysis. Study comprised of a prostate cancer patient cohort with organ confined primary tumors (N=338) presenting following post-surgery features: 53 (15.7%) metastatic progression, 124 (36.7%) biochemical recurrence (BCR), and 161 (47.6%) no progression after more than ten years of follow-up after radical prostatectomy. Index tumor region for each case was scraped from representative 10-m sections of formalin-fixed paraffin embedded (FFPE)-whole-mounted prostatectomy specimens and processed for PRISM-SRM analysis. Results: Overall, PRISM-SRM analysis of the FFPE tissue samples enabled the detection of 42 (80.8%) out of 52 biomarker candidates; in comparison regular LC-SRM without the front-end chromatographic enrichment could detect only 21 (40.4%) of these candidates at the protein level. Kruskal-Wallis testing was used for statistical evaluation of the PRISM-SRM results and comparison of relative protein levels between the “no progression”, BCR and “metastatic progression” groups. Several prostate differentiation/androgen receptor signaling related proteins (FOLH1, PSA and NCOA) and tumor progression-related proteins (TGFB1, CCND1 and SPRC) had significantly different expression levels between the three groups, and showed initial promise in predicting progression to invasive cancer, BCR and metastasis. Conclusion: Ultra-sensitive targeted proteomics can be used to select and verify performance of early prognostic markers based on the analysis of prostatectomy specimens. The top performing markers appeared able to predict progression from organ confined cancer to BCR and metastasis. Citation Format: Yuqian Gao, Yi-Ting Wang, Hui Wang, Denise Young, Jennifer Cullen, Yingjie Song, Yongmei Chen, Athena Schepmoes, Gyorgy Petrovics, Thomas Fillmore, Tujin Shi, Wei-Jun Qian, Richard Smith, Sudhir Srivastava, Jacob Kagan, Albert Dobi, Inger Rosner, Karin Rodland, Isabell Sesterhenn, Shiv Srivastava, Tao Liu. Identification of candidate biomarkers for aggressive prostate cancer using targeted proteomics and FFPE tissue samples with outcomes data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3165.

Prognostic and therapeutic implications of lysyl oxidase and cyclooxygenase 2 expressions in epithelial ovarian carcinoma

Background: New prognostic and predictive biomarkers for better choice and improving the current therapies for ovarian cancer are greatly needed. This study aimed to investigate the prognostic and therapeutic importance of lysyl oxidase (LOX) and cyclooxygenase 2 (COX2) expressions in epithelial ovarian carcinoma. Methods: We performed immunohistochemical analysis on formalin-fixed paraffin sections of epithelial ovarian tumors. The association between their expressions in epithelial ovarian carcinoma (EOC) with the survival as well as response to chemotherapy was analyzed. Results: The frequency of the nuclear expression of LOX and cytoplasmic expression of COX2 was significantly higher in malignant tumors than in benign and borderline tumors. Also, there were statistically significant relationships between pathological grades and both LOX and COX2 positivity in EOC being at the higher end for poorly differentiated tumors. Both LOX and COX2 expressions were also correlated significantly with higher tumor stage. Overexpression of either LOX or COX2 in EOC was significantly associated with poor survival. Moreover, LOX and COX2 positive expressions in EOC were associated with poor response to chemotherapy. Conclusions: The increased expressions of LOX and COX2 in EOC are associated with several adverse clinicopathologic parameters, including reduced survival and chemotherapy resistance thus suggesting a role for such biomarkers in disease progression.

Dimethyl Oxalyl Glycine a Prolyl Hydroxylase Inhibitor Decreases Early Gastrointestinal Gvhd Via Two Independent Pathways of Apoptosis after Allogeneic Hematopoietic Cell Transplantation

Introduction: Allogeneic hematopoietic cell transplantation (HCT) is increasingly utilized in the treatment of hematologic malignant and non-malignant diseases. Therapy-related toxicity and graft versus host disease (GVHD) remain major challenges as they significantly contribute to treatment related morbidity and mortality. Prolyl hydroxylase inhibitors (PHI) interfere with signaling cascades of inflammation and cell death. Their beneficial use in experimental models of ulcerative colitis and lung allograft rejection resulted in the hypothesis, that use of the PHI dimethyl oxalyl glycine (DMOG) reduces GVHD after murine allogeneic HCT. We already showed that DMOG treatment of allogeneic recipients resulted in significantly improved survival by day +50 compared to allogeneic controls, and was associated with significant decrease in organ pathology and proinflammatory cytokines (data not shown). We now expanded our study to better understand the underlying mechanisms for this observation.Methods: Acute GVHD was induced in lethally irradiated BALB/c mice followed by the infusion of 5 million bone marrow cells mixed with 4 million splenocytes from allogeneic C57BL/6 donors. DMOG dissolved in PBS was given at 8mg per mice intraperitoneally while controls received PBS only. Animals were monitored for clinical GVHD and survival and analyzed at day +7 and at day +50 post HCT. Formalin-fixed paraffin sections were processed for immunohistochemistry with CD3, Hif-1 alpha, or FADD pSer191 antibodies. Western blot analysis was performed after CD3/CD28 antibody activation of T cells to see the effect of DMOG treatment on Hif-1 α expression. Effect of DMOG on apoptosis and CD137 expression was analyzed by flow cytometry.Results: Our results demonstrate that the protective effect of DMOG is mainly caused by two independent pathways of apoptosis. DMOG treatment of allogeneic recipients resulted in decreased T cells infiltration (p=0.0534) and (Fas associated death domain) FADDpSer191 level (p<0.05) in small intestine sections at day +50. Decreased FADD pSer191 indicates decreased TNF-a induced epithelial cell apoptosis. This was associated with increased Hif1- α expression at day+7 (p<0.05) as well day+50 (p<0.05) as shown by histochemical analysis of small intestine sections. DMOG treatment of activated T cells showed increased expression of CD137 (p<0.005) and Annexin V FITC (p<0.005) staining suggesting that DMOG subjects T cells to activation induced cell death (AICD) via induced CD137 expression. Microscopic scanning of DMOG treated T cells showed condensed chromatin along with numerous fragmented nuclei, indicative of apoptotic cell. In vitro proliferative T cell responses were suppressed (p<0.05) in the presence of DMOG in a dose-dependent manner along with increase in Hif1- α level (p<0.05) as analyzed by western blot.Conclusion: Our data demonstrates protective effects of DMOG on the GI tract early after allogeneic HCT which is associated with improved survival and reduced lung injury at later time point. DMOG treatment results in the induction of CD137 on T cells subjecting them to AICD and represses FADD level in epithelial cells regulating their apoptosis. Taken together, DMOG treatment reduces GVHD via a two pronged mechanism, possibly allowing for novel strategies to prevent and treat this deleterious disease. DisclosuresPalaniyandi:Pharmacyclics: Research Funding; Takeda Pharmaceutical International, Inc.: Research Funding; JAZZ: Research Funding.

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network.

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to <20 µm as formalin fixed paraffin section on glass slides or <500 µm for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples >500 µm using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth >150 µm with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

High Resolution 3D Imaging of the Human Pancreas Neuro-insular Network

Using traditional histological methods, researchers are hampered in their ability to image whole tissues or organs in large-scale 3D. Histological sections are generally limited to <20 µm as formalin fixed paraffin section on glass slides or <500 µm for free-floating fixed sections. Therefore, extensive efforts are required for serial sectioning and large-scale image reconstruction methods to recreate 3D for samples >500 µm using traditional methods. In addition, light scatters from macromolecules within tissues, particularly lipids, prevents imaging to a depth >150 µm with most confocal microscopes. To reduce light scatter and to allow for deep tissue imaging using simple confocal microscopy, various optical clearing methods have been developed that are relevant for rodent and human tissue samples fixed by immersion. Several methods are related and use protein crosslinking with acrylamide and tissue clearing with sodium dodecyl sulfate (SDS). Other optical clearing techniques used various solvents though each modification had various advantages and disadvantages. Here, an optimized passive optical clearing method is described for studies of the human pancreas innervation and specifically for interrogation of the innervation of human islets.

Rapid immunocytochemistry for the detection of cytokeratin and vimentin: assessment of its diagnostic value in neoplastic diseases of dogs.

Immunocytochemistry (ICC) is an advanced diagnostic technique used in the field of veterinary cytology. We recently developed a rapid ICC method for the detection of cytokeratin and vimentin in dogs, which helps to determine whether tumor cells are of epithelial or nonepithelial origin. However, the diagnostic value of this rapid ICC method in neoplastic diseases of dogs has not been assessed yet. The aim of the present study was to assess the diagnostic accuracy of rapid ICC compared to standard immunohistochemistry (IHC). Air-dried smear samples and formalin-fixed paraffin sections were prepared from tumors excised from dogs (n=30). Immunosignals for cytokeratin and vimentin were detected in smear samples by rapid ICC, and in paraffin sections by standard IHC. Signals in smear samples detected by rapid ICC were compared with positive staining in paraffin sections detected by standard IHC and analyzed for statistical significance (kappa statistic). Rapid ICC detected specific immunosignals in 25/30 cases (83.3%), and nonspecific signals were detected in 5/30 cases. Statistical analysis revealed fair agreement in epithelial tumors (n=16) with cytokeratin (κ=0.236) and vimentin (κ=0.294). In nonepithelial tumors (n=14), almost perfect agreement was demonstrated with cytokeratin (κ=0.857) and vimentin (κ=0.857). The rapid ICC method can be a useful tool for the diagnostic cytology of neoplastic tissues in dogs.

PWE-039 The Interaction between The Hypoxia Inducible Factor-1Α (HIF-1Α) and ERBB Signalling Pathways in Colorectal Liver Metastases (CRLM)

Introduction Tumour hypoxia is associated with reduced survival by activating multiple molecular pathways such as, but not exclusively, HIF-1α and mTOR/AKT/PI3K pathways. It is also known to cause overexpression of Epidermal Growth Factor (EGFR)/HER1 in human cancer. HER2 (neu) signalling increases the rate of Hypoxia-Inducible Factor 1α (HIF-1α) synthesis. Overexpression of Erbb3/HER3 and Erbb4/HER4 is associated with a higher recurrence rate of stage 2 and 3 colorectal cancer. The aim of this study was to analyse the relationship between the expression of HIF-1α and members of the ErbB pathway in CRLM utilising a novel proteomic based collaborative Enzyme Enhanced Reactive (CEER) immunoassay. Methods Protein lysates of from fresh frozen biopsies and formalin fixed paraffin sections of CRLM from 18 patients who had not undergone neoadjuvant chemotherapy prior to a potentially curative hepatectomy for CRLM were analysed. When multiple metastases were present biopsies and sections were obtained from the largest metastasis. CEER immunoassay was performed on the protein lysates to quantify the phosphorylated and non-phosphorylated forms of receptors; ErbB family receptors (HER1/EGFR, HER2/c-neu, HER3, HER4) and cMET, and phosphorylated downstream ErbB pathway factors AKT, PRAS, RSP6, ERK, and RSK, and CK. Immunostaining was performed on formalin-fixed-paraffin-embedded CRLM sections for HIF-1α. ErbB pathway factor quantification was segregated according to the HIF-1α expression status; HIF-1α +ve and HIF-1α -ve Results HIF-1α was expressed in 15 (83%) of tumours. There was no significant difference between the expression of phosphorylated ‘receptors’, and phosphorylated forms AKT, PRAS, RSP6, ERK, MEK, and PI3K between the HIF-1α +ve and HIF-1α –ve groups. HER3 and IGF1R were higher; (median µm: range; 87 [74–344] vs 222 [60–404]) and (115 [85–206] vs 186 [90–285]) respectively in the HIF-1α +ve group, HER1, HER2, PI3K and CK levels were lower; (108 [40–316] vs 36 [22–46]), (698 [523–870] vs 481 [288–709]), (5338 [3334–6689] vs 2719 [1790–4193]), (5383 [1826–9799] vs 2719 [1789–4193]) respectively in the HIF-1α +ve group. Although a trend was observed, the difference did not reach statistical significance. Conclusion HIF-1α may have a role in the overexpression of HER3 and IGF1R overexpression in CRLM. More research is needed to establish this interrelationship which would help in molecular based therapeutics for CRLM. Disclosure of Interest None Declared

Downregulation of IDH2 exacerbates the malignant progression of osteosarcoma cells via increased NF-κB and MMP-9 activation.

Isocitrate dehydrogenase 2 (IDH2) is a mitochondrial NADP-dependent isocitrate dehydrogenase. It is considered to be a novel tumor suppressor in several types of tumors. However, the role and related mechanism of IDH2 in osteosarcoma remain unknown. The expression and significance of IDH2 were investigated by immunohistochemistry in formalin-fixed paraffin sections from 44 osteosarcoma patients. IDH2 was downregulated via lentiviral vector‑mediated RNA interference (RNAi) in the Saos-2 and MG-63 human osteosarcoma cell lines. The effect of IDH2 downregulation on human osteosarcoma was studied in vitro by MTT, flow cytometry and invasion assays. Nuclear factor-κB (NF-κB) and matrix metalloproteinase-9 (MMP-9) assays were also used to study the likely molecular mechanism of IDH2 downregulation on the malignant progression of osteosarcoma cells. The results revealed that the expression of IDH2 was inversely correlated with pathological grade and metastasis in osteosarcoma. IDH2 downregulation promoted a pro-proliferative effect on the Saos-2 and MG-63 osteosarcoma cell lines. IDH2 downregulation accelerated cell cycle progression from S to G2/M phase. The pro-proliferative effect induced by IDH2 downregulation may be ascribed to increased NF-κB activity via IκBα phosphorylation. The invasive activity of osteosarcoma cells was also significantly promoted by IDH2 downregulation and may result from elevated MMP-9 activity. In conclusion, IDH2 downregulation may exacerbate malignant progression via increased NF-κB and MMP-9 activity and may implicate the potential biological importance of IDH2 targeting in osteosarcoma cells. Downregulation of IDH2 exacerbates the malignant progression of osteosarcoma cells via increased NF-κB and MMP-9 activation.

Diagnosis of Amyloidosis Subtype By Laser-Capture Microdissection (LCM) and Tandem Mass Spectrometry (MS/MS) Proteomic Analysis

AimCorrect identification of the protein that is causing amyloidosis is crucial for clinical management. Current standard laboratory methods have limited ability to detect the full range of amyloid forming proteins. We assessed the diagnostic value of LCM-MS/MS, which combines specific sampling of amyloid deposits by LCM with protein identification by MS/MS.MethodsBiopsy specimens were referred to the Princess Alexandra Hospital Amyloidosis Centre. For all specimens, 10µm sections of formalin-fixed paraffin embedded tissue were stained with Congo Red using a standard technique. LCM was performed using an Arcturus XT instrument with an infrared capture laser. Proteins were extracted with FFPE Protein Extraction Solution (Agilent Technologies), digested with trypsin and peptides were analysed by nano-liquid chromatography-coupled MS/MS using an Agilent Chip CUBE-QTOF. Database searching was performed using Spectrum Mill (Agilent) with the NCBInr human protein database.ResultsBiopsies were received on 136 patients: there was insufficient tissue in the block in 7, repeat LCM was required in15 cases and no amyloid forming protein was identified in 8. In 121/136 (89%) an amyloid forming protein was identified. Proteins identified included immunoglobulin light chain (localised amyloid n=25, systemic AL n=45), immunoglobulin heavy chain (AH n=6), transthyretin (senile amyloid n=25, hereditary ATTR n=6), serum amyloid A (AA n=7), fibrinogen alpha chain (AFib n=2), LECT2 (ALect2 n=2), TGFb (corneal lattice amyloid n=1) and semenogelin (seminal vesicle amyloid n=2). It was not infrequent, particularly in cases of localised amyloidosis (>80% of cases), for smaller amounts of other amyloid forming proteins to be present especially immunoglobulins, transthyretin and ApoA1 (Table 1). This suggests that these inherently amyloidogenic proteins are capable of integrating within the amyloid deposit. An amyloid proteomic signature as previously defined by the Mayo Clinic (at least two of SAP, ApoE and ApoA4; Haematologica 2014;99(7):1239) was present in 76% of cases and was more likely to be found if larger amounts of amyloid could be dissected (p=0.0001).In terms of clinical impact, amyloid typing by immunohistochemical stains had been attempted in 87 cases and reported as diagnostic in 39. Five of these were subsequently revealed by proteomic analysis to be incorrect. Overall, the clinical diagnosis of amyloid subtype was altered by proteomic analysis in 24% of cases.Conclusion.LCMMS/MS identifies an amyloid forming protein in ~90% of clinical biopsy samples. Amyloid deposits often contain small amounts of other amyloid forming proteins which may reflect not just contamination but co-deposition of fibrils due to a shared beta pleated sheet conformation. Because of this, results need to be interpreted in the context of full clinical information to enable correct diagnosis of amyloid subtype.Table 1LCM-MS/MS resultsAmyloidosis subtypeAL-lambdaAL-kappaAHLocalised lambdaLocalised kappaATTRwtATTRmutAANumber of subtype cases321351692567Amyloid forming proteinsLambda light chain321612Kappa light chain41325941Ig heavy chain8566413Transthyretin133256ApoA12373SAA7Fibrinogen alpha chain2121Lysozyme111Cases with low levels of 2nd amyloid forming protein34%46%40%81%89%20%17%57%Amyloid associated proteinsApoE211221671965SAP1681362464ApoA4231011682262Amyloid proteomic signature63%77%0%100%78%92%100%57% DisclosuresMollee:Onyx: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Effect of Gualou Xiebai Banxia decoction combined with Xuefu Zhuyu decoction on myocardial apoptosis and relevant protein expressions in miniature swine phlegm and blood stasis type coronary heart disease model

There have been very few studies on the effect of Gualou Xiebai Banxia decoction combined with Xuefu Zhuyu decoction in inhibiting apoptosis in myocardial ischemial injury caused by coronary heart disease. In this experiment, Gualou Xiebai Banxia decoction combined with-Xuefu Zhuyu decoction were used to intervene the miniature swine phlegm and blood stasis type coronary heart disease model, in order to observe the effect of the combined prescription on the myocardial apoptosis and the expressions of Bcl-2, Bax, Caspase-3, Caspase-9 in the model. Totally 15 Chinese experimental miniature swine were adopted and randomly divided into the control group, the model group and the phlegm and stasis-treating group. The model group and the stasis-treating group were fed with high fat diets for two weeks, intervened with the coronary artery injury and then given drugs and high fat diets for eight weeks. The control group was fed with ordinary diets for 10 weeks, without the coronary artery injury. After the experiment, myocardia at the juncture of infracted areas were collected and made into formalin-fixed paraffin sections. The TDT-mediate dUTP nick end labeling (TUNEL) assay was used to detect the myocardial apoptosis. The immunohistochemistry (IHC) technique was applied to detect Bcl-2, Bax, Caspase-3, Caspase-9 levels in myocardial tissues. According to the findings, the apoptosis indexes (AI) for the control group, the model group and the phlegm and stasis-treating group were 0.92%, 27.68%, 17.28%, respectively. The AI of the phlegm and stasis-treating group was significantly lower than that of the model group (P < 0.01). Compared with the model group, the phlegm and stasis-treating group showed significantly higher Bcl-2 protein expression (P < 0.01) and lower Bax, Caspase-3 and Caspase-9 protein expressions (P < 0.01). In conclusion, Gualou Xiebai Banxia decoction combined with Xuefu Zhuyu decoction have a significant protective effect against the myocardial apoptosis in miniature swine phlegm and blood stasis type coronary heart disease model.

9 Matrix remodeling in head and neck squamous cell carcinomais

Extracellular matrix (ECM) remodeling is an inherent characteristic of invasive head and neck squamous cell carcinoma (HNSCC). We investigated the regulation of gene expression, synthesis and activation of ECM components and matrix remodeling enzymes in a co-culture model of head and neck squamous cell carcinoma (HNSCC) and in HNSCC tissue. Periodontal ligament fibroblasts (PDL) and SCC-25 HNSCC cells were co-cultured for seven days, followed by mRNA and protein expression analysis of ECM components, ECM receptors, matrix metalloproteinases (MMPs) and proteinase inhibitors. The activity of MMPs was analyzed using gelatinase zymography. MMP-9, fibronectin and “antiadhesion proteins” were investigated by immunohistochemistry in HNSCC tissue. In co-cultured fibroblasts, in addition to fibronectin (FN), the “antiadhesion proteins” tenascin C (TSC), thrombospondin 1 (TSP1) and latent TGF-beta binding protein 2 (LTBP2) were upregulated. In lower levels: FN, TSC and TSP1 were also produced by the tumor cells. In co-cultured fibroblasts, alpha 1 and 5 and beta 1 integrins, in co-cultured tumor cells alpha 3 and 5 integrins were upregulated. MMP-1 was upregulated in both co-cultured tumor cells and fibroblasts, MMP-2 in co-cultured fibroblasts, and MMP-9 in co-cultured tumor cells. MMP inhibitors were mainly produced in fibroblasts. The activation of MMP-2 required the co-culture with tumor cells. MMP-9 was exclusively produced in tumor cells. In sections of formalin-fixed paraffin embedded HNSCC tissue, FN and LTBP2 were detected in fibroblastic stroma surrounding tumor cell nests; TSP1 was detected both in tumor cell nests and in the surrounding fibroblastic stroma. Extended MMP-9 immunohistochemical staining was found in 27 of 280 HNSCC tissue samples, in a subpopulation of cells in tumor cell nests. Fibroblast – tumor cell communication is a major pathway of matrix remodeling in HNSCC tumor tissue. Carcinoma-associated fibroblasts produce new matrix elements as TSP1, TSC and LTBP2, both tumor cells and fibroblasts upregulate integrin receptors, and they also share tasks in synthesis and activation of matrix remodeling enzymes. Intensive matrix re-organization takes place at the borders of tumor cell nests.